Liquid chromatography provides a means for separating the contents of a liquid sample based upon the polarity, solubility and mobility of the individual components. Within the past twenty years, high performance liquid chromatography (HPLC) has revolutionized the world of analytical chemistry by providing highly efficient columns and packing materials which allow for the separation of very closely related components of a liquid sample.
One drawback to either conventional liquid chromatography or HPLC has been that all samples must be completely dissolved before being applied to the column for separation. Any particulate material which reaches the sorbent or solid phase packing material may adversely effect the flow rate through the column and separation power of the packing and ultimately result in repacking the column or in the case of HPLC, disposal of the column and packing.
To prevent the inadvertent destruction of the packing material, filters or frits or guard columns have been placed at the head of the column which are designed to trap particulate matter which may inadvertently be injected. However, these filters and guard columns are known to clog necessitating extensive cleaning procedures or replacement. Thus, samples which contain partially dissolved material such as cell residue, mycelia, tissue homogenate, debris, etc., must be clarified before being placed on the column.
Several attempts have been made to circumvent the problems presented by the presence of particulate matter in a sample.
For example, simple filtration is often effective if the components of interest can be completely dissolved in the solvent used to prepare the sample. However, simple filtration is not an option when the analyte cannot be completely dissolved away from insoluble materials, such as biological samples where cell fragments may retain some of the material of interest.
In order to overcome this problem, techniques have been developed which involve multiple extraction of the samples and subsequent evaporation of the extract to dryness, followed by redissolution of the sample and finally separation of the resultant particle free solution using a solid phase extraction cartridge or by direct injection onto a chromatography column. The vigorous pre-treatment of the sample, to assure that any undissolved matter is first removed, is time consuming and costly.
A standard solid phase extraction cartridge, such as those manufactured and sold commercially by Applied Separations, Inc., Diazem.TM. Corporation, EM Science (a division of EM Industries, Inc.), Keystone Scientific, Inc., United Chemical Technologies, Inc., Waters Corporation, Whatman.RTM. Inc., Worldwide Monitoring.RTM., YMC, Inc. and many others is commonly used in this procedure. These devices consist of a column having a porous frit or filter which divides the column into a top and bottom section, where the bottom section contains a sorbent such as silica particles which are chemically reacted so that their surface contains various organic functional groups such as phenyl, octyl, octadecyl, or organic or inorganic polymers. When using these cartridges, a solubilized sample substantially free from particulate matters that could clog the top frit is added to the top section of the column and the liquid is allowed to flow either by gravity, under vacuum or by externally applied gas pressure, through the porous frit and then through the sorbent. As the sample passes through the sorbent, certain solutes are either absorbed or adsorbed onto the sorbent and retained. Various solvents are then used to elute the sorbed material in a sequential manner such that separation based upon polarity, solubility and mobility of the various compounds may be effected. The eluted samples may often be applied to an HPLC column or examined with other analytical techniques with or without further concentration.
While the combined use of the liquid extraction/redissolution technique and solid phase extraction cartridges is effective, it suffers from three major problems. First, the sample applied to the column must be substantially free from particulate matter capable of clogging the frit. Second, there is no assurance that all material in the original sample has been fully extracted from the insoluble matter by means of batchwise extraction methods. Third, the extractions, evaporation and redissolution of the sample introduces several inherent errors which must be considered when interpreting the final results.
Thus, what is needed is a simple economic device and procedure which provides for the separation of components, based on their polarity, solubility and mobility extracted from the original biological sample, according to the principles of counter-current extraction wherein the entire contents of the sample, both liquid and solid, can be treated directly, without clogging the column and with minimum inherent error.